Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters.

Parenchymal liver cells were isolated from human liver pieces of surgical waste as well as from rat livers. DNA synthetic activity was measured after different times in primary culture by [3H]thymidine incorporation and autoradiography. Labeling of control cultures of human hepatocytes at densities between 8,000 and 15,000 cells/cm2 was very low (0.4 to 1.3%). Human recombinant epidermal growth factor increased labeling 2- to 4-fold (P less than 0.01). Treatment with known inducers of liver growth in rats, namely, cyproterone acetate, alpha-hexachlorocyclohexane, nafenopin, phenobarbital, and rifampicin did not increase the number of labeled human liver cells. In some of the experiments, a 24-h exposure to the chemicals of rat or human hepatocytes was followed by a 24-h treatment with epidermal growth factor (EGF). In rat hepatocytes, incorporation rates were significantly increased. Cyproterone acetate and EGF acted in an additive manner, alpha-hexachlorocyclohexane and EGF were clearly overadditive, and phenobarbital had little effect. In human hepatocytes, little alteration in labeling indices was found; in some cases labeling was, rather, found to be lower than in cultures treated with EGF alone. These results show that human hepatocytes cultured in vitro are sensitive to stimulation of DNA synthesis by EGF; they differ from rat hepatocytes in their response to some drugs which show liver growth-promoting activity in rodents.     

The induction of unscheduled DNA synthesis by antihistamines in primary hepatocyte cultures

The autoradiographic identification of chemically-induced, unscheduled DNA synthesis in primary cultures of adult rat hepatocytes is currently being validated as a predictive test for mutagens/carcinogens. Of 8 antihistaminic drugs tested, 2, pyrilamine maleate and tripelennamine HCl, were positive for unscheduled DNA synthesis. Further investigation of the mutagenic/carcinogenic potential of these compounds in alternate test systems is suggested.     

Induction of DNA fragmentation and DNA repair synthesis in human and rat hepatocytes by diethylstilbestrol

The synthetic estrogen diethylstilbestrol (DES), a known human carcinogen, was examined for cytotoxicity, and the induction of DNA damage and repair in primary cultures of human and rat hepatocytes. In both species concentrations of DES ranging from 5.6 to 18 micrograms/ml constantly produced reduction of cell viability and DNA fragmentation in dose-related amounts. However, large individual quantitative differences in the sensitivity to the cytotoxic and DNA-damaging activities of DES were observed among cultures derived from the 5 human donors. DES capability of eliciting DNA-excision repair was weak but statistically significant in both human and rat hepatocytes. Taken as a whole these results contribute to support the hypothesis of a genotoxic mechanism in DES-induced carcinogenesis.     

Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures.

Unscheduled DNA synthesis was observed in primary rat liver cell cultures treated with members of five different classes of chemical procarcinogens requiring enzymatic activation as well as with a direct-acting carcinogen. In total, ten carcinogens and one related analog not commonly accepted as carcinogenic were active, while one weak carcinogen and four noncarcinogens were inactive. The production of unscheduled DNA synthesis by this spectrum of chemical carcinogens indicates that these cultures have substantially retained the metabolic capability of liver for activating diverse procarcinogens. Thus, such cultures may be useful for detecting the ability of chemicals to interact with DNA and, thereby, assigning them priority for consideration as potential cancer-causing agents.     

Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures

Cyproterone acetate (CPA), an active component of certain contraceptiveand antiandrogenic drugs, has been shown recently to induceDNA repair synthesis in rat hepatocytes in vitro. In the presentstudy we examined whether CPA can cause the formation of DNAadducts detectable by the ³²P-postlabeling technique in hepaticcells in vitro and in vivo. Incubation of primary cultures ofhepatocytes from male Wistar rats with CPA resulted in the occurrenceof radioactive spots in the radiochromatograms of ³²P-postlabeledDNA digests indicating the formation of two DNA adducts (‘A’and ‘B’). At 30 µM CPA, the highest concentrationtested,     

Stimulation of DNA synthesis in primary rat hepatocyte cultures by liver tumor promoters: interactions with other growth factors.

Mechanism(s) of tumor promotion in liver by xenobiotics such as hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) and phenobarbital (PB) are not understood in detail although growth-stimulatory effects may be significant in their action. As a basis for studying mechanisms of growth control by liver tumor promoters, effects of xenobiotics on DNA synthesis have been examined in primary cultures of normal rat hepatocytes, maintained under fully-defined conditions. The xenobiotics alone were relatively ineffective but they exhibited synergism with epidermal growth factor (EGF), insulin and dexamethasone in stimulating DNA synthesis and were effective in moderate-to-low density cultures but not in confluent monolayers. Under conditions optimized for HCH or pregnenolone 16 alpha-carbonitrile (PCN) (i.e. subconfluent cultures exposed to insulin, EGF and dexamethasone), HCH, PCN, DDT or PB caused a transient stimulation of DNA synthesis, apparent after 2 days in culture. This probably reflected earlier entry of hepatocytes to S-phase. HCH was shown to increase total DNA, total numbers of nuclei and numbers of cells undergoing mitosis per culture. In optimized conditions, HCH or PCN were about additive with norepinephrine, dialyzed serum or pyruvate or with a small effect of tri-iodothyronine in stimulating DNA synthesis. Although conditions optimal for HCH or PCN were not necessarily optimal for detecting growth-stimulatory effects of other xenobiotics or steroids, these culture conditions were shown to support stimulation of DNA synthesis by a variety of known liver tumor promoters including barbiturates, estrogens, progestins, peroxisomal proliferators and bile acids. Several compounds known not to promote liver carcinogenesis failed to stimulate DNA synthesis in similar hepatocyte cultures.     

Induction of ornithine decarboxylase and DNA synthesis in rat stomach mucosa by methylglyoxal

Administration of methylglyoxal at doses of 300–600 mg/kgbody weight by gastric tube to male F344 rats induced 100-foldincrease in ornithine decarboxylase activity (formation of 682pmol CO₂/30 min/mg protein) within 7 h, 26-fold increase inDNA synthesis (incorporation of 17 800 d.p.m. of [³H]thymidine/µgDNA) within 16 h, 16-fold increase in the labeling index ofS-phase cells (increase from 1.7 to 26.5) within 16 h, and apparentunscheduled DNA synthesis within 2 h in the glandular stomachmucosa. These results suggest that methylglyoxal has potentialpromoter activity and may also have initiating activity in carcinogenesisin the glandular stomach.     

Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture

Iron-overload diseases frequently develop hepatocellular carcinoma. The genotoxic mechanism whereby iron is involved in hepatocarcinogenesis might involve an oxidative process via the intermediate production of reactive oxygen species. This was presently investigated by examining kinetics of formation and repair of DNA base lesions in primary rat hepatocyte cultures supplemented with the iron chelate, ferric nitrilotriacetate Fe-NTA (10 and 100 microM). Seven DNA base oxidation products have been identified in DNA extracts by gas chromatography- mass spectrometry, which showed a predominance of oxidized-purines (8- oxo-guanine, xanthine, fapy-adenine, 2-oxo-adenine) above oxidized pyrimidines (5-OHMe-uracil, 5-OH-uracil, 5-OH-cytosine) in control cultures. All these DNA oxidation products revealed a significant dose- dependent increase at 4 to 48 h after Fe-NTA supplementation, among which fapy-adenine showed the highest increase and 5-OH-cytosine was the least prominent. Involvement of iron in this oxidative process was established by a correlation between extent in DNA oxidation and intracellular level of toxic low molecular weight iron. DNA excision- repair activity was estimated by release of DNA oxidation products in culture medium. All the seven DNA oxidation products were detected in the medium of control cultures and showed basal repair activity. This DNA repair activity was increased in a time- and dose-dependent fashion with Fe-NTA. Oxidized-pyrimidines, among which was 5-OHMe-Uracil, were preferentially repaired, which explains the low levels detected in oxidized DNA. Since oxidized bases substantially differed from one another in terms of excision rates from cellular DNA, specific excision- repair enzymes might be involved. Our findings, however, demonstrate that even though DNA repair pathways were activated in iron-loaded hepatocyte cultures, these processes were not stimulated enough to prevent an accumulation of highly mutagenic DNA oxidative products in genomic DNA. The resulting genotoxic effect of Fe-NTA might be relevant in understanding the hepatocarcinogenic evolution of iron-overload diseases.      

Induction of DNA replication and cell growth in rat liver by obstructive jaundice

Obstructive jaundice, produced by ligating the common bile duct, induced a transient DNA replication followed by cell proliferation in rat liver. At 48 h after the operation, DNA polymerase alpha activity started to increase and reached its maximum level (more than twice the control) at day 4. At day 7, the enzyme level had decreased to the control level. Pulse-labeling experiment using radioactive thymidine showed that the rate of DNA synthesis increased approximately 2.5-fold in the same pattern as that of DNA polymerase alpha. The mitotic index in hepatocytes also increased 10-fold at day 4 and then decreased. The proliferation of liver cells induced by obstructive jaundice mimics the regeneration of partially hepatectomized liver, although the response was slightly delayed and the proliferation was transient.     

Inductions of ornithine decarboxylase and replicative DNA synthesis but not DNA single strand scission or unscheduled DNA synthesis in the pyloric mucosa of rat stomach by catechol

The possible tumor-promoting and genotoxic activities of catechol were examined. Administration of catechol by gastric intubation at doses of 10 to 90 mg/kg body weight to male F344 rats induced up to 19-fold increase in ornithine decarboxylase activity with a maximum after 8 h and up to 8-fold increase in replicative DNA synthesis with a maximum after 24 h in the pyloric mucosa of the stomach. These results suggest that catechol has tumor-promoting activity in the pyloric mucosa of rat stomach. However, its administration at doses of 37.5 to 90 mg/kg body weight did not induce DNA single strand scission in the pyloric mucosa as determined by the alkaline elution method after 2 and 6 h or unscheduled DNA synthesis examined after 2 and 12 h.     

Unscheduled DNA synthesis caused by norethindrone and related contraceptive steroids in short-term male rat hepatocyte cultures

The genotoxic potential of oral contraceptive steroids suchas norethindrone was investigated in short-term rat hepatocytecultures by measurement of unscheduled DNA synthesis. Norethindronecaused a small dose-dependent increase in unscheduled DNA synthesisin male rat hepatocytes as judged by the incorporation of [methyl-³H]thymidineinto DNA. This was assessed either by liquid scintillation countingfollowing isolation of DNA or by autoradiography. No increasein unscheduled DNA synthesis could be detected in female rathepatocytes treated with norethindrone. Pre-treatment of malerats with phenobarbitone prior to hepatocyte preparation decreasedthe norethindrone mediated unscheduled DNA synthesis relativeto control hepatocyte cultures while 3-methylcholanthrene pre-treatmenthad little effect. Unscheduled DNA synthesis in norethindronetreated control male rat hepatocytes was reduced by the mixedfunction oxidase inhibitors SKF 525A or metyrapone. In 24- or52-hourold hepatocyte cultures in which the cytochrome P-450content was lower than in freshly prepared cells, or in a hepatocyte-derivedcell line lacking cytochrome P-450, unscheduled DNA synthesisdue to norethindrone was either decreased or abolished. Structureactivity studies showed that only steroids containing a 17-ethynylsubstituent caused an increase in unscheduled DNA synthesis.     

Induction of DNA repair synthesis in isolated rat hepatocytes by 5-diazouracil and other DNA damaging compounds

5-Diazouracil, which has been recommended in cancer chemotherapy, induced DNA repair synthesis in isolated rat hepatocytes. Repair synthesis was determined by means of the bromodeoxyuridine (BrdUrd)-CsCl-gradient centrifugation method. The validity of this technique was demonstrated using various DNA damaging compounds.     

Wy-14,643, a peroxisome proliferator, inhibits compensative cell proliferation and hepatocyte growth factor mRNA expression in the rat liver

Previously, we found that a peroxisome proliferator significantly reduced hepatic and plasma hepatocyte growth factor (HGF) levels in male F-344 rats, and that the growth of preneoplastic or neoplastic cells induced by this peroxisome proliferator was markedly inhibited by HGF. Here, we examined the effects of [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643), a peroxisome proliferator, on cell proliferation and HGF mRNA levels in the liver of rats after stimulation of compensative cell proliferation. After 2 weeks of treatment with Wy-14,643, hepatic DNA synthesis caused by partial hepatectomy was decreased by 50% compared with untreated controls. DNA synthesis was maintained at the same reduced level for up to 10 weeks. During this period, hepatic HGF mRNA level was also much lower in Wy-14,643-treated rats than untreated controls. Therefore Wy-14,643, a peroxisome proliferator, would inhibit the growth of normal hepatocytes, and then produce an advantageous circumstance for the selective growth of neoplastic or preneoplastic cells.     

Calcium chloride inhibits stimulation of replicative DNA synthesis by sodium chloride in the pyloric mucosa of rat stomach

Calcium chloride (C_aCl₂ inhibits stimulation of replicativeDNA synthesis (RDS) induced in the pyloric mucosa of male Fischer344 rats by sodium chloride (NaCl), which is a tumor promoterin the glandular stomach. Administration of 1 ml of 3.3 M NaClby gastric intubation induced a maximal 15-fold increase inRDS in the pyloric mucosa by 17 h; this had returned to thecontrol level by 48 h. Administration of 1 ml of 20–400mM CaCl₂ 1 h before administration of NaCl resulted in a 60–100%inhibition of the increase in RDS within 4–48 h; the inhibitionwas dose-dependent. The 400 mM level of CaCl₂ also decreasedthe histological damage to surface epithelial cells inducedby NaCl. These results suggest that calcium ion acts as an anti-tumorpromoter in stomach carcinogenesis.     

Stimulation of DNA synthesis and c-fos mRNA expression in primary rat hepatocytes by estrogens

The mechanism(s) of tumour promotion in liver by estrogens is not well understood although growth stimulation is known to be one important element of their action. As a basis for studying mechanisms of growth control by estrogens, effects of both natural and synthetic estrogens on DNA synthesis and protooncogene c-fos mRNA expression were examined in primary cultures of normal rat hepatocytes. 17beta-Estradiol (E(2)) alone was stimulatory and exhibited dramatic synergism with epidermal growth factor (EGF) in stimulating DNA synthesis. All estrogens tested (natural, synthetic, steroidal and non-steroidal) exhibited an ability to stimulate hepatocyte DNA synthesis. This appears to correlate with their ability to induce c-fos mRNA expression. In contrast to a non-estrogenic liver tumour promoter, phenobarbital, insulin is not permissive for the growth-stimulatory action of E(2). Dexamethasone, which is required for stimulation of DNA synthesis by the non-estrogenic tumour promoter alpha-hexachlorocyclohexane and tetradecanoylphorbol acetate, completely blocked E(2)-stimulated DNA synthesis. Such differential requirements for auxiliary factors suggests that estrogen and other non-estrogenic liver tumour promoters act via distinct mechanisms in stimulating hepatocyte DNA synthesis. E(2) alone had no effect, but when in combination with EGF significantly induced c-fos mRNA expression at early times in culture (maximal at 10 h in culture). Such findings, coupled with the observations that (i) E(2) and EGF were synergistic in growth stimulation, (ii) estrogen receptor levels are higher at early times in culture and (iii) the growth-stimulatory ability of E(2) is limited to 4-24 h in culture, support the notion that in hepatocytes E(2) acts via the estrogen receptor to transactivate c-fos expression (an interaction with EGF), which ultimately culminates in enhanced DNA synthesis. Dexamethasone did not block E(2)-induced c-fos gene expression, suggesting that it acts in a pathway(s) distal to activation of fos gene expression. The possible inhibitory mechanisms of action of dexamethasone on E(2)-stimulated DNA synthesis are discussed.     

Differential effect of adriamycin on DNA replicative and repair synthesis in cultured neonatal rat cardiac cells.

The effect of the potent antitumor antiobiotic Adriamycin (ADM) on DNA replication and unscheduled DNA synthesis in cultured rat cardiac cells was investigated. Autoradiography and [3H]thymidine incorporation studies were carried out on parallel cultures. DNA replication was depressed for up to 6 days following a 3-hr pulse of ADM administration. An ADM concentration of 1 microgram/ml which was effective in reducing replicative DNA synthesis by as much as 75% did not reduce the ability of cardiac cells to repair UV-damaged DNA. However, cells exposed to higher ADM concentrations failed to undergo significant UV-induced repair. In the absence of UV treatment, ADM did not stimulate unscheduled DNA synthesis. To account for the differential response of the cardiac cell cultures to replicate and repair DNA, we propose that ADM exerts a localized effect on DNA synthesis covering a region proximal to its primary intercalation site.     

Induction of rat kidney ornithine decarboxylase by nicotinamide without a concomitant increase in DNA synthesis

Four hours after an i.p. injection of 500 mg/kg nicotinamide,there was a decrease in kidney ODC activity, followed by a substantialincrease by 24 h. Exposing rats to 0, 0.67, 6.7 and 30 mM nicotinamidein their drinking water for 7 and 28 days also resulted in astatistically significant increase in kidney ODC activity, butthe rates of DNA synthesis were unaffected, as measured by incorporationof [³H]thymidine into DNA and % labeled proximal tubule nuclei;the total amount of DNA/kidney was also unaffected. The resultsof this investigation demonstrate that nicotinamide, a knownrenal tumor promoter, was able to induce a significant increasein ODC activity in the rat kidney without a concomitant increasein DNA synthesis, suggesting that early stimulation of ODC isassociated with tumor promotion even in the absence of effectson DNA replication.     

Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene

The purpose of this study was to examine the induction of unscheduledDNA synthesis (UDS) by the potent hepatocarcinogen technicalgrade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) usingthe in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344rats were treated by gavage and hepatocytes were isolated byliver perfusion and cultured with [³H]thymidine. UDS was measuredby quantitative autoradiography as net grains/nucleus (NG);5 NG was considered positive. Controls consistently had –3 to – 6 NG. A dose-related increase in UDS was observed12 h after treatment, with 200 mg/kg tgDNT producing 26 NG.A 50-fold increase in the number of cells in S-phase was observedat 48 h after treatment. This increase in S-phase cells couldbe suppressed in the presence of 10–20 mM hydroxyurea(HU), while the same levels of HU did not affect the level ofUDS at 12 h after treatment. 2, 4-DNT produced only a weak response,in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatmentof female rats with tgDNT yielded only modest increases in UDSand DNA replication relative to males. These results are consistentwith the carcinogenicity studies and indicate that tgDNT isa potent genotoxic agent, with 2, 6-DNT contributing the majorportion of the effect.